International Journal of Pharmaceutics (v.443, #1-2)

Study of the stability of proteoliposomes as vehicles for vaccines against Neisseria meningitidis based on recombinant porin complexes by Paula Freixeiro; Ernesto Diéguez-Casal; Liliana Costoya; Begoña Seijo; Carlos M. Ferreirós; María Teresa Criado; Sandra Sánchez (1-8).
Although effective against epidemic serogroup B Neisseria meningitidis strains, vaccines based on outer membrane vesicles continue to present important limitations, and great efforts are currently being focused in the development of a variety of new vaccine candidates and in the reformulation of currently existing ones. In this work, three N. meningitidis proteins, the PorA and PorB porins and the RmpM protein, were cloned, purified and incorporated into liposomes to build defined systems. The ability of proteoliposomes to allow the refolding porin complexes, and their stability during storage at 4 °C and after lyophilization in presence of two cryoprotection agents, glucose and trehalose, were evaluated. This approach allowed to mimic the porin complexes present in natural OMVs, reducing the content of hypervariable protein PorA. During storage at 4 °C, our systems showed some changes in the morphology and aggregation after three months, while after lyophilization the systems maintained their properties during the whole nine months of storage checked, with glucose allowing the best preservation of the antigenic properties of the proteins in the proteoliposomes.
Keywords: Liposome stability; Lyophilization; Protein refolding; Vaccine; Neisseria meningitidis;

Characterization of the pharmacokinetics of a liposomal formulation of eribulin mesylate (E7389) in mice by Yanke Yu; Christopher DesJardins; Phil Saxton; George Lai; Edgar Schuck; Y. Nancy Wong (9-16).
Eribulin mesylate (E7389), a tubulin and microtubule inhibitor, has been approved to treat metastatic breast cancer in certain patient populations. A liposomal formulation of E7389, E7389-LF, aims to increase the therapeutic profile of E7389. As determining the free drug concentration is crucial for the assessment of efficacy and toxicity of liposomal drug, in this study, an ultracentrifugation method coupled with LC–MS/MS was developed to separate the free E7389 from liposomal and protein bound E7389. The pharmacokinetics of the free E7389 after dosing either E7389 or E7389-LF was characterized. The concentration ratio of E7389 in ultracentrifuged mice plasma (UCM) vs E7389 in plasma after a 2 mg/kg i.v. of E7389 ranged from 54.19% to 65.41%, which was similar to the free fraction in the mouse plasma. The respective concentration ratio of E7389 in UCM vs E7389 in plasma after a 2 mg/kg i.v. of E7389-LF ranged from 0.07% to 0.59%, and the exposure, expressed as AUC, of UCM/plasma ratio was determined to be 0.2%. Pharmacokinetic modeling was performed to estimate the release kinetics of E7389 from E7389-LF, and the release was best described by a first order rate constant k rel 0.078 h−1. Sensitivity analysis demonstrated that further decrease the release rate constant by adjusting liposome formulation would lead to decreased C max and much longer half-life of UCM E7389, which might result in better efficacy and lower toxicity.
Keywords: E7389; E7389-LF; Ultracentrifugation; Pharmacokinetics; Modeling; Sensitivity analysis;

Prolongation of time interval between doses could eliminate accelerated blood clearance phenomenon induced by pegylated liposomal topotecan by ChunLei Li; Xi Zhao; YaJuan Wang; HanYu Yang; HaiXia Li; Huan Li; Wei Tian; Jian Yang; JingXia Cui (17-25).
The prolongation of time interval between doses from 1 week (A) to 4 weeks (B) could eliminate the accelerated blood clearance phenomenon induced by pegylated liposomal topotecan.Repeated injection of pegylated liposomes could elicit the disappearance of long-circulating characteristic, referred to as “accelerated blood clearance phenomenon.” ABC phenomenon typically occurs when entrapped drugs are not cytotoxic, but recently it was reported that multiple doses of pegylated liposomal topotecan, a cytotoxic drug, could also induce the phenomenon in rats. To reveal whether the phenomenon could be induced in dogs and the effect of time interval between doses on the magnitude of ABC, pLT was repeatedly injected into beagle dogs with a time interval of 7, 21 and 28 days. The anti-PEG Ig M levels were detected using ELISA. It was found that ABC phenomenon could be induced in dogs by pLT. Inter-individual difference in anti-PEG antibody production could be observed, and antibody levels were directly correlated with the magnitude of ABC. Furthermore, time interval between doses had marked effect on the magnitude of ABC phenomenon. When the time interval was prolonged from 1 week to 4 weeks, ABC phenomenon could be eliminated. By comparing the pharmacokinetic profiles of lipid vesicles and entrapped topotecan, it was found that “empty pegylated vesicles” be formed in circulation, which might be responsible for the occurrence of ABC phenomenon.
Keywords: Pegylated liposomes; Repeated injection; Accelerated blood clearance; Time interval; Topotecan;

Valsartan was loaded into statistically optimized nanoethosomes for enhanced transdermal delivery and characterized for various parameters including entrapment efficiency, vesicles shape, size, size distribution, skin permeation and in vivo pharmacokinetic study. Nanoethosomes of valsartan provides better flux, reasonable entrapment efficiency, and more effectiveness for transdermal delivery as compared to conventional liposomes. Pharmacokinetic study of nanoethosomal system showed a significant increase in bioavailability as compared to oral suspension. It can be concluded that nanoethosomes is a potentially useful carrier for improved transdermal delivery of valsartan for the management of hypertension.The aim of the current investigation is to develop and statistically optimize nanoethosomes for transdermal valsartan delivery. Box-Behnken experimental design was applied for optimization of nanoethosomes. The Independent variables were phospholipids 90G (X 1), ethanol (X 2), valsartan (X 3) and sonication time (X 4) while entrapment efficiency (Y 1), vesicle size (Y 2) and flux (Y 3) were the dependent variables. The optimized formulation obtained was then tested in rats for an in vivo pharmacokinetic study. Results indicate that the nanoethosomes of valsartan provides better flux, reasonable entrapment efficiency, more effectiveness for transdermal delivery as compared to rigid liposomes. Optimized nanoethosomal formulation with mean particle size is 103 ± 5.0 nm showed 89.34 ± 2.54% entrapment efficiency and achieved mean transdermal flux 801.36 ± 21.45 μg/cm2/h. Nanoethosomes proved significantly superior in terms of, amount of drug permeated in the skin, with an enhancement ratio of 43.38 ± 1.37 when compared to rigid liposomes. Confocal laser scanning microscopy revealed an enhanced permeation of Rhodamine-Red loaded nanoethosomes to the deeper layers of the skin as compared to conventional liposomes. In vivo pharmacokinetic study of nanoethosomal transdermal therapeutic system showed a significant increase in bioavailability (3.03 times) compared with oral suspension of valsartan. Our results suggest that nanoethosomes are an efficient carrier for transdermal delivery of valsartan.
Keywords: Transdermal; Ethosomes; Pharmacokinetics; Valsartan; Hypertension;

Red blood cells as innovative antigen carrier to induce specific immune tolerance by Magali Cremel; Nathalie Guérin; Françoise Horand; Alice Banz; Yann Godfrin (39-49).
The route of administration, the dose of antigen as well as the type of antigen-presenting cells (APCs) targeted are important factors to induce immune tolerance. Despite encouraging results obtained in animal models, intravenous injection of soluble antigen is unsuccessful in human clinical trials on autoimmune disease due to inefficient antigen delivery. To improve antigen delivery, we used mouse red blood cells (RBCs) as antigen vehicles to specifically target APCs which are responsible for removal of senescent RBCs after phagocytosis. In this study, we demonstrated that antigen-delivery by RBCs induced a strong decrease in the humoral response compared with the ovalbumin (OVA) free form in mice. In addition, OVA-loaded RBC treated with [bis(sulphosuccinimidyl)] suberate (BS3), a chemical compound known to enhance RBC phagocytosis, induced an inhibition of antigen-specific T cell responses and an increase in the percentage of regulatory T cells. The state of tolerance induced is long lasting, antigen-specific and sufficiently robust to withstand immunization with antigen mixed with cholera toxin adjuvant. This RBC strategy, which does not abolish the immune system, constitutes an attractive approach for induction of tolerance compared to systemic immunosuppressant therapies already in use.
Keywords: Red blood cell carrier; Tolerance induction; Immunotherapy; Cellular immunology; Antibodies;

Preparation and evaluation of raloxifene-loaded solid dispersion nanoparticle by spray-drying technique without an organic solvent by Tuan Hiep Tran; Bijay K. Poudel; Nirmal Marasini; Sang-Cheol Chi; Han-Gon Choi; Chul Soon Yong; Jong Oh Kim (50-57).
The aim of this study was to improve the physicochemical properties and bioavailability of a poorly water-soluble drug, raloxifene by solid dispersion (SD) nanoparticles using the spray-drying technique. These spray-dried SD nanoparticles were prepared with raloxifene (RXF), polyvinylpyrrolidone (PVP) and Tween 20 in water. Reconstitution of optimized RXF-loaded SD nanoparticles in pH 1.2 medium showed a mean particle size of approximately 180 nm. X-ray diffraction and differential scanning calorimetry indicated that RXF existed in an amorphous form within spray-dried nanoparticles. The optimized formulation showed an enhanced dissolution rate of RXF at pH 1.2, 4.0, 6.8 and distilled water as compared to pure RXF powder. The improved dissolution of raloxifene from spray-dried SD nanoparticles appeared to be well correlated with enhanced oral bioavailability of raloxifene in rats. Furthermore, the pharmacokinetic parameters of the spray-dried SD nanoparticles showed increased AUC0–∞ and C max of RXF by approximately 3.3-fold and 2.3-fold, respectively. These results suggest that the preparation of RXF-SD nanoparticles using the spray drying technique without organic solvents might be a promising approach for improving the oral bioavailability of RXF.
Keywords: Raloxifene; Solubility; Bioavailability; Spray drying; Nanoparticles;

A majority of quantitative structure–permeability relationships (QSPeRs) predict the permeability coefficient (k p ) of compounds topically applied as infinite, saturated doses from water vehicles. Alternate delivery vehicles and other experimental variables are rarely incorporated in such models. This research presents the development and statistical validation of QSPeR models that incorporate the effects of penetrant, vehicle, and experimental conditions such as dose volume (finite/infinite), and saturation level (saturated/unsaturated). A composite parameter, a mixture factor (MF), was also included to account for the physicochemical properties of the compound/vehicle mixture components. The resultant models effectively described skin flux and absorption, identifying the summation of hydrogen bond acidity and basicity, excess molar refractivity, dose volume, saturation level, and vehicle as the most prominent factors influencing flux values. The main factors influencing absorption values were the summation of hydrogen bond basicity, dipolarity/polarizability, the McGowan characteristic volume, dose volume, saturation level, and vehicle. The same MF (inverse of the melting point) was considered suitable to describe both flux and absorption. For endpoints involving skin deposition, log propylene glycol solubility was a more suitable MF. Such models show potential for use in drug delivery and toxicology research, specifically in assessing percutaneous absorption data collected under different experimental conditions.
Keywords: Quantitative structure–permeability relationships (QSPeRs); Experimental variables; Mixture factor; Steady-state flux; Percutaneous absorption;

PLGA microparticles with zero-order release of the labile anti-Parkinson drug apomorphine by C. Regnier-Delplace; O. Thillaye du Boullay; F. Siepmann; B. Martin-Vaca; N. Degrave; P. Demonchaux; O. Jentzer; D. Bourissou; J. Siepmann (68-79).
The treatment of patients suffering from advanced Parkinson's disease is highly challenging, because the efficacy of the “gold standard” levodopa declines with time. Co-administration of the dopamine receptor agonist apomorphine is beneficial, but difficult due to the poor oral bioavailability and short half-life of this drug. In order to overcome these restrictions, PLGA-based microparticles allowing for controlled parenteral delivery of this morphine derivative were prepared using (solid-in-)oil-in-water extraction/evaporation techniques. Particular attention was paid to minimize spontaneous oxidation of the labile drug and to optimize the key features of the microparticles, including encapsulation efficiency, initial burst release and particle size. Various formulation and processing parameters were adjusted in this respect. The systems were thoroughly characterized using SEM, EDX, DSC, laser diffraction, headspace-GC as well as in vitro drug release measurements in agitated flasks and flow-through cells. Importantly, apomorphine could effectively be protected against degradation during microparticle preparation and within the delivery systems upon exposure to phosphate buffer pH 7.4 (containing 0.2% ascorbic acid) at 37 °C: 90% intact drug was released at a constant rate during about 10 d.
Keywords: Controlled release; Microparticle; PLGA; Apomorphine; Parkinson's disease;

The purpose of this study was to establish a novel nontoxic disulfide bond reducing method for lipophilic drug-loaded albumin nanoparticle preparation and make a systematic investigation on this method. Cysteine (Cys) was used to break the disulfide bond of albumin and introduce the self-assembly of drug and albumin. Paclitaxel (PTX) and bovine serum albumin (BSA) were selected to be the model lipophilic drug and albumin. The particle formation dynamics, influencing factors and formation mechanisms were investigated by determining the characteristics of particles including the particle size and yield. Nanoparticles with diameter of 50–400 nm and drug loading efficiency up to 18.3% were prepared successfully. pH 7–8 was suitable for nanoparticle preparation. Temperature, BSA concentration and Cys concentration had positive effects on the particle size and yield. When PTX added was less than the maximal amount of PTX that could bind to BSA, particles with a spherical structure could be formed; otherwise nanoparticles with a core–shell structure could be formed. This novel nontoxic disulfide bond reducing method provides a common and safe method for preparing various kinds of albumin-based nanocarriers for a wide range of applications, from drug (especially the lipophilic drug) delivery to diagnosis of disease.
Keywords: Disulfide bond reducing method; Nanoparticle; Cysteine; Paclitaxel; Bovine serum albumin;

Preparation and characterization of PLGA microspheres by the electrospraying method for delivering simvastatin for bone regeneration by Subrata Deb Nath; Sora Son; Alexandar Sadiasa; Young Ki Min; Byong Taek Lee (87-94).
Microparticles formulated from poly (D,L-lactic-co-glycolide) (PLGA), a biodegradable polymer, have been investigated extensively as a drug delivery system. In this study, solid tiny PLGA microspheres were fabricated using the electrospraying method. PLGA polymer was dissolved in dichloromethane (DCM), and the solution was electrosprayed. The electrospraying conditions were adjusted so that the stream ejected from the needle was divided into spheres instead of continuous fibers or irregular-shaped particles. Several experiments were carried out using the PLGA-DCM source solution with different amounts of simvastatin (SIM), a drug that enhances bone regeneration, to understand this drug delivery system. The surface morphology and microstructure of the microspheres formed were characterized by scanning electron microscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, and differential scanning calorimetry. The in vitro experiments on drug loading and drug release behavior of the microspheres suggested a drug encapsulation efficacy >90%. The drug was continuously released from the microspheres for >3 weeks. Other experiments, such as MTT, cell attachment and proliferation and reverse transcription-polymerase chain reaction showed good biocompatibility of the electrosprayed PLGA microspheres, which increased in the presence of SIM. Thus, electrosprayed PLGA microspheres have potential as a drug delivery system and application in bone tissue engineering.
Keywords: PLGA; Simvastatin; Microspheres; Drug delivery system; Electrospraying;

Solid dispersions of varying weight ratios compositions of the nonionic drug, griseofulvin and the hydrophilic, anionic polymer, hydroxylpropylmethyl cellulose acetate succinate, have been prepared by ball milling and the resulting samples characterized using a combination of Fourier transform infra-red spectroscopy, X-ray powder diffraction and differential scanning calorimetry. The results suggest that griseofulvin forms hydrogen bonds with the hydroxylpropylmethyl cellulose acetate succinate polymer when prepared in the form of a solid dispersion but not when prepared in a physical mixture of the same composition. As anticipated, the actual measured glass transition temperature of the solid dispersions displayed a linear relationship between that predicted using the Gordon–Taylor and Fox equations assuming ideal mixing, but interestingly only at griseofulvin contents less than 50 wt%. At griseofulvin concentrations greater than this, the measured glass transition temperature of the solid dispersions was almost constant. Furthermore, the crystalline content of the solid dispersions, as determined by differential scanning calorimetry and X-ray powder diffraction followed a similar trend in that the crystalline content significantly decreased at ratios less than 50 wt% of griseofulvin. When the physical mixtures of griseofulvin and the hydroxylpropylmethyl cellulose acetate succinate polymer were analyzed using the Flory–Huggins model, a negative free energy of mixing with an interaction parameter of −0.23 were obtained. Taken together these results suggest that anionic hydrophilic hydroxylpropylmethyl cellulose acetate succinate polymer is a good solvent for crystalline nonionic griseofulvin with the solubility of griseofulvin in the solid dispersion being was estimated to be within the range 40–50 wt%. Below this solubility limit, the amorphous drug exists as amorphous glassy solution while above these values the system is supersaturated and glassy suspension and solution may coexist.
Keywords: Amorphous solid dispersions; Glass transition; Crystallinity; Hydrogen bonds; Flory–Huggins interaction parameter;

Biocompatible gemcitabine-based nanomedicine engineered by Flow Focusing® for efficient antitumor activity by Lucía Martín-Banderas; Eva Sáez-Fernández; Mª Ángeles Holgado; Mª Matilde Durán-Lobato; José C. Prados; Consolación Melguizo; José L. Arias (103-109).
We investigated the incorporation of gemcitabine into a colloidal carrier based on the biodegradable and biocompatible poly(d,l-lactide-co-glycolide) (PLGA) to optimize its anticancer activity. Two synthesis techniques (double emulsion/solvent evaporation, and Flow Focusing®) were compared in terms of particle geometry, electrophoretic properties (surface charge), gemcitabine vehiculization capabilities (drug loading and release), blood compatibility, and in vitro antitumor activity. To the best of our knowledge, the second formulation methodology (Flow Focusing®) has never been applied to the synthesis of gemcitabine-loaded PLGA particles. With the aim of achieving the finest (nano)formulation, experimental parameters associated to these preparation procedures were analyzed. The electrokinetics of the particles suggested that the chemotherapy agent was incorporated into the polymeric matrix. Blood compatibility was demonstrated in vitro. Flow Focusing® led to a more appropriate geometry, higher gemcitabine loading and a sustained release profile. In addition, the cytotoxicity of gemcitabine-loaded particles prepared by Flow Focusing® was tested in MCF-7 human breast adenocarcinoma cells, showing significantly greater antitumor activity compared to the free drug and to the gemcitabine-loaded particles synthesized by double emulsion/solvent evaporation. Thus, it has been identified the more adequate formulation conditions in the engineering of gemcitabine-loaded PLGA nanoparticles for the effective treatment of tumours.
Keywords: Antitumor drug; Double emulsion/solvent evaporation; Flow Focusing®; Gemcitabine; Nanoparticles; Poly(d,l-lactide-co-glycolide);

Controlled drug release system based on cyclodextrin-conjugated poly(lactic acid)-b-poly(ethylene glycol) micelles by Qin He; Wei Wu; Kemao Xiu; Qian Zhang; Fujian Xu; Jianshu Li (110-119).
Cyclodextrin-conjugated poly(lactic acid)-b-poly(ethylene glycol) (β-CD-PLA-mPEG), a well-defined amphiphilic copolymer, was synthesized by controlled ring-open copolymerization and click coupling reaction, in order to obtain a biocompatible drug delivery system with controlled release profiles. The β-CD-PLA-mPEG copolymer could self-assemble in aqueous solution to form micelles with a mean particle size of 173.4 nm, which will decrease to 159.2 nm after loaded with a kind of hydrophobic drug (indomethacin, IND). The IND-loaded β-CD-PLA-mPEG micelles show spherical shape within the nano-size scale under TEM imaging. Compared with that formed by PLA-mPEG, the micelles formed by β-CD-PLA-mPEG copolymer present higher drug loading efficiency and controlled release profile of IND, especially in the control of its initial burst release. Meanwhile, β-CD-PLA-mPEG copolymer exhibits low toxicity to cells. The micelles formed by β-CD-PLA-mPEG copolymer could be a promising controlled release system for various hydrophobic drugs.
Keywords: Controlled release; Indomethacin; Cyclodextrin-conjugates; Micelle; Cytotoxicity;

Effect of unsaturated menthol analogues on the in vitro penetration of 5-fluorouracil through rat skin by Yang Chen; Jian Wang; Dongmei Cun; Manli Wang; Juan Jiang; Honglei Xi; Hongxia Cui; Yongnan Xu; Maosheng Cheng; Liang Fang (120-127).
To explore the structure–activity relationship for terpenes as transdermal penetration enhancers, unsaturated menthol analogues were synthesized in our study, including p-menth-1-en-3-ol (Compd 1), p-menth-4-en-3-ol (Compd 2), p-menth-4(8)-en-3-ol (Compd 3) and p-menth-8-en-3-ol (Compd 4). Their enhancing activity on the penetration of 5-fluorouracil through rat skin was evaluated by in vitro experiments. Attenuated total reflection-Fourier transform infrared spectroscopy, molecular modeling and transepidermal water loss (TEWL) were introduced to investigate the enhancer induced alteration in different skin lipid domains. The results indicated that Compd 3 achieved the highest enhancement ability with an enhancement ratio of 3.08. Other analogues were less effective than Compd 3, and no significant difference was found between them and menthol. Treatment of rat skin with these enhancers did not produce any shift in the stretching vibration of the methylene in hydrophobic lipid chains, but significantly improved the polar pathway across the rat skin as suggested by the increased TEWL. Molecular modeling results suggested that polar head groups of the skin lipids provided the main binding site for enhancer action. These findings indicated that the studied compounds enhanced drug transport by interacting with the polar domain of the skin lipid, instead of by affecting the arrangement of the hydrophobic chains.
Keywords: Transdermal; Absorption enhancer; Unsaturated; Menthol analogue; Mechanism; Structure–activity relationship;

Inhibition of skin inflammation in mice by diclofenac in vesicular carriers: Liposomes, ethosomes and PEVs by Carla Caddeo; Octavio Diez Sales; Donatella Valenti; Amparo Ruiz Saurí; Anna Maria Fadda; Maria Manconi (128-136).
Protocol for treatment of TPA-induced inflammation in mouse skin.Diclofenac-loaded phospholipid vesicles, namely conventional liposomes, ethosomes and PEVs (penetration enhancer-containing vesicles) were developed and their efficacy in TPA (phorbol ester) induced skin inflammation was examined. Vesicles were made from a cheap and unpurified mixture of phospholipids and diclofenac sodium; Transcutol® P and propylene glycol were added to obtain PEVs, and ethanol to produce ethosomes. The structure and lamellar organization of the vesicle bilayer were investigated by transmission electron microscopy and small and wide angle X-ray scattering, as well as the main physico-chemical features. The formulations, along with a diclofenac solution and commercial Voltaren Emulgel®, were tested in a comparative trial for anti-inflammatory efficacy on TPA-treated mice dorsal skin.Vesicles were around 100 nm, negatively charged, able to encapsulate diclofenac in good yields, and disclosed different lamellarity, as a function of the formulation composition. Vesicular formulations promoted drug accumulation and reduced the permeation. Administration of vesicular diclofenac on TPA-inflamed skin resulted in marked attenuation of oedema and leucocyte infiltration, especially using PEVs. Histology confirmed the effectiveness of vesicles, since they provided an amelioration of the tissual damage induced by TPA.The proposed approach based on vesicular nanocarriers may hold promising therapeutic value for treating a variety of inflammatory skin disorders.
Keywords: Vesicles; Skin inflammation; Oedema; Myeloperoxidase; Histology;

The aim of this study was to develop a spray dried submicrometer powder formulation suitable for the excipient enhanced growth (EEG) application. Combination particles were prepared using the Buchi Nano spray dryer B-90. A number of spray drying and formulation variables were investigated with the aims of producing dry powder formulations that were readily dispersed upon aerosolization and maximizing the fraction of submicrometer particles. Albuterol sulfate, mannitol, l-leucine, and poloxamer 188 were selected as a model drug, hygroscopic excipient, dispersibility enhancer and surfactant, respectively. Formulations were assessed by scanning electron microscopy and aerosol performance following aerosolization using an Aerolizer® dry powder inhaler (DPI). In vitro drug deposition was studied using a realistic mouth–throat (MT) model. Based on the in vitro aerosolization results, the best performing submicrometer powder formulation consisted of albuterol sulfate, mannitol, l-leucine and poloxamer 188 in a ratio of 30:48:20:2, containing 0.5% solids in a water:ethanol (80:20%, v/v) solution which was spray dried at 70 °C. The submicrometer particle fraction (FPF1 μm/ED) of this final formulation was 28.3% with more than 80% of the capsule contents being emitted during aerosolization. This formulation also showed 4.1% MT deposition. The developed combination formulation delivered a powder aerosol developed for the EEG application with high dispersion efficiency and low MT deposition from a convenient DPI device platform.
Keywords: Excipient enhanced growth (EEG); Spray drying; Vibrating mesh; Hygroscopic aerosol; Dry powder inhaler;

Pharmaceutical and safety considerations of tablet crushing in patients undergoing enteral intubation by Damien Salmon; Elisa Pont; Hélène Chevallard; Elhadji Diouf; Mamadou-Lamine Tall; Christine Pivot; Fabrice Pirot (146-153).
Medication in patients undergoing enteral intubation addresses various challenging issues considering safety and treatment efficiency. Ideally, other routes of administration (i.e. intravenous or intramuscular routes) or especially dedicated formulations should be used. However, in absence of liquid dosage form, tablets or pills must be crushed and suspended in a vehicle before administration. The administration of oral dosage forms by enteral tube is usually performed by the nursing staff facing (i) pharmaceutical relevance of crushing, (ii) loss and concomitant aero-contamination of drug substance, (iii) drug–nutriment interactions and (iv) enteral feeding tube clogging. In the present study, different combinations of either open or confined crushing and suspending protocols were compared by taking into account the crushing yield, the stability and granulometry of the solid oral form suspension and finally the extend of aerosol contamination during crushing and suspending. All protocols exhibited comparable crushing efficiency and suspending properties, but significantly higher aerosolisation of tablet particles was observed in both open crushing and suspending protocol. Therefore, both confined crushing and suspending protocol constitutes an efficient, time saving and safe alternative to the absence of available liquid dosage form for intubated patients.
Keywords: Crushing; Enteral nutrition; Medication; Tablet; Aerosolisation; Suspension;

A non-invasive and external stimulus-driven local drug delivery system (DDS) based on titania nanotube (TNT) arrays loaded with drug encapsulated polymeric micelles as drug carriers and ultrasound generator is described. Ultrasound waves (USW) generated by a pulsating sonication probe (Sonotrode) in phosphate buffered saline (PBS) at pH 7.2 as the medium for transmitting pressure waves, were used to release drug-loaded nano-carriers from the TNT arrays. It was demonstrated that a very rapid release in pulsatile mode can be achieved, controlled by several parameters on the ultrasonic generator. This includes pulse length, time, amplitude and power intensity. By optimization of these parameters, an immediate drug-micelles release of 100% that spans a desirable time of 5–50 min was achieved. It was shown that stimulated release can be generated and reproduced at any time throughout the TNT-Ti implant life, suggesting considerable potential of this approach as a feasible and tunable ultrasound-mediated drug delivery system in situ via drug-releasing implants. It is expected that this concept can be translated from an in vitro to in vivo regime for therapeutic applications using drug-releasing implants in orthopedic and coronary stents.
Keywords: Local drug delivery; Titania nanotubes; Stimulated drug-micelles release; Drug carrier; Polymeric micelles; Ultrasound;

High surface area polypyrrole scaffolds for tunable drug delivery by Manisha Sharma; Geoffrey I.N. Waterhouse; Samuel W.C. Loader; Sanjay Garg; Darren Svirskis (163-168).
Intrinsically conducting polymers such as polypyrrole (PPy) are viable platforms for efficient drug delivery, where release rates can be tuned by external electrical stimulus. In this study, the successful fabrication of 3-dimensionally ordered macroporous PPy inverse opal thin films is described, and the viability of such films for controlled drug release evaluated in vitro. The PPy inverse opal thin films were obtained by electropolymerization of PPy through the interstitial voids of a colloidal crystal template composed of poly(methyl methacrylate) colloids of diameter ∼430 nm. Chemical etching of the template yielded macroporous PPy inverse opal scaffolds. The model drug risperidone was loaded into the PPy inverse opal films, and then entrapped by electropolymerization of a non-porous PPy overlayer. The morphology and chemical composition of the PPy scaffolds were evaluated by SEM and FTIR spectroscopy, respectively. The high surface area PPy inverse opal scaffolds exhibited enhanced drug loading and releasing capabilities compared to conventional non-porous PPy films. Drug release profiles could be modified by applying electrical stimulus, which caused actuation of the porous polypyrrole films. The proposed delivery system may find use as an implantable device where drug release can be electrically tuned according to patient requirements.
Keywords: Polypyrrole; Inverse opal; Drug delivery; Tunable drug release;

Oral insulin delivery in rats by nanoparticles prepared with non-toxic solvents by Angela Viehof; Lucie Javot; Arnaud Béduneau; Yann Pellequer; Alf Lamprecht (169-174).
Nanoparticles (NPs) have shown a certain potential to overcome the drawbacks of oral peptide delivery in the gastrointestinal tract such as low peptide stability and permeability. The preparation of insulin loaded NPs was carried out with Eudragit® RL or RS dissolved in different non-toxic polyethylene glycol (PEG) derivatives. The use of these non-toxic solvents allowed the design of an one step NP preparation method where insulin retained its full biological activity as it was proven in vitro and in vivo. The insulin trapping NPs were in a size range of around 150–250 nm and exhibited a pH-dependent release. The type of solvent did not distinctly influence the particle properties or insulin stability but modified significantly the performance in vivo in rats, NPs prepared with glycofurol led to a bioavailability of F  = 1.4 ± 1.0% after oral administration while NPs prepared with PEG 300 were hardly efficient (F  = 0.3 ± 0.5%). In all cases t max was shifted to 2 h compared to 1 h after subcutaneous insulin solution. In general, we believe that the method presented here is a promising way to encapsulate sensitive drugs, especially for the production of peptide loaded NPs.
Keywords: Nanoparticles; Non-toxic solvent; Glycofurol; Polyethylene glycol; Insulin; Oral drug delivery;

Curcumin loaded polymeric micelles inhibit breast tumor growth and spontaneous pulmonary metastasis by Lei Liu; Lu Sun; Qinjie Wu; Wenhao Guo; Ling Li; YiShan Chen; Yuchen Li; Changyang Gong; Zhiyong Qian; Yuquan Wei (175-182).
Curcumin loaded polymeric micelles (Cur-M) were prepared using a one-step solid dispersion method to overcome poor water solubility of Cur and to meet the requirement of intravenous administration. The Cur-M were more effective in inhibiting tumor growth and spontaneous pulmonary metastasis in the 4T1 breast tumor model.This work aims to develop curcumin (Cur) loaded biodegradable self-assembled polymeric micelles (Cur-M) to overcome poor water solubility of Cur and to meet the requirement of intravenous administration. Cur-M were prepared by solid dispersion method, which was simple and easy to scale up. Cur-M had a small particle size of 28.2 ± 1.8 nm and polydisperse index (PDI) of 0.136 ± 0.050, and drug loading and encapsulation efficiency of Cur-M were 14.84 ± 0.11% and 98.91 ± 0.70%, respectively. Besides, in vitro release profile showed a significant difference between rapid release of free Cur and much slower and sustained release of Cur-M. Cytotoxicity study showed that the encapsulated Cur remained its potent anti-tumor effect. Furthermore, Cur-M were more effective in inhibiting tumor growth and spontaneous pulmonary metastasis in subcutaneous 4T1 breast tumor model, and prolonged survival of tumor-bearing mice. In addition, immunofluorescent and immunohistochemical studies also showed that tumors of Cur-M-treated mice had more apoptosis cells, fewer microvessels, and fewer proliferation-positive cells. In conclusion, polymeric micelles encapsulating Cur were developed with enhanced anti-tumor and anti-metastasis activity on breast tumor, and Cur-M is excellent water-based formulation of Cur which may serve as a candidate for breast cancer therapy.
Keywords: Curcumin; Biodegradable; Micelle; Breast tumor; Spontaneous pulmonary metastasis;

Control over hygroscopic growth of saline aqueous aerosol using Pluronic polymer additives by Allen E. Haddrell; Graham Hargreaves; James F. Davies; Jonathan P. Reid (183-192).
The hygroscopic properties of an aerosol originating from a nebulizer solution can affect the extent of peripheral deposition within the respiratory tract, which in turn affects drug efficacy of drugs delivered to the lungs. Thus, the ability to tailor the degree and rate of hygroscopic growth of an aerosol produced by a nebulizer through modification of the formulation would serve to improve drug efficacy through targeted lung deposition. In this study, the kinetic and thermodynamic hygroscopic properties of sodium chloride aerosol mixed with commercially available Pluronic polymers, specifically F77 and F127, are reported using three complementary single aerosol analysis techniques, specifically aerosol optical tweezers, a double ring electrodynamic balance and a concentric cylinder electrodynamic balance. The F77 polymer is shown to have a predictable effect on the hygroscopic properties of the aerosol: the ability of the droplet to uptake water from the air depends on the solute weight percent of sodium chloride present in a linear dose dependant manner. Unlike the smaller F77, a non-linear relationship was observed for the larger molecular weight F127 polymer, with significant suppression of hygroscopic growth (>50% by mass) for solution aerosol containing even only 1 wt% of the polymer and 99 wt% sodium chloride. The suppression of growth is shown to be consistent with the formation of mixed phase aerosol particles containing hydrophilic inorganic rich domains and hydrophobic polymer rich domains that sequester some of the inorganic component, with the two phases responding to changes in relative humidity independently. This independence of coupling with the gas phase is apparent in both the equilibrium state and the kinetics of water evaporation/condensation. By starting with a saline nebulizer solution with a concentration of F127 ∼10−2  mM, a 12% reduction in the radius of all aerosol produced at a relative humidity (RH) of 84% is possible. The difference in diameter is RH dependent, and may be much greater at higher humidities. These findings suggest that the addition of μM concentrations of larger Pluronic polymers to nebulizer formulations may greatly reduce the size of aerosols produced.
Keywords: Aerosols; Hygroscopic growth; Polymers; Drug delivery to the lungs;

Organic solvent vapor effects on phase transition of α and β tegafur upon grinding with solvent additives by Raitis Bobrovs; Olga Saveļjeva; Agnese Kapace; Zane Plauka; Andris Actiņš (193-198).
Solvent effects on α tegafur (5-fluoro-1-(tetrahydro-2-furyl)uracil) phase transition to β tegafur during grinding with solvent additive, as well as phase transition in samples exposed to 95% relative solvent vapor pressure has been studied in this research. Samples containing 0.5% and 0.1% of β tegafur in α and β tegafur mixture, as well as samples of pure α tegafur were ground with different solvent additives, and conversion degrees depending on the solvent were determined using PXRD method. Samples with α and β tegafur weight fraction of 1:1 were exposed to 95% relative solvent vapor pressure, and phase transition rates were determined. Solubility of α tegafur, solvent sorption and desorption behavior on α and β tegafur have been examined.It was found that the conversion degree of α tegafur to β tegafur mainly depends on solubility of α tegafur in the relevant solvent, and the conversion degree to β tegafur is higher in such solvents, where solubility of α tegafur is higher. The samples ground in a ball mill with solvent additive had a trend of phase transition dynamics from α tegafur to β tegafur similar to the samples exposed to 95% relative solvent vapor pressure.
Keywords: Grinding; Phase transition; Polymorphism; Tegafur; Sorption; Desorption; Solubility; X-ray powder diffractometry;

Drug–polymer intermolecular interactions in hot-melt extruded solid dispersions by Mohammed Maniruzzaman; David J. Morgan; Andrew P. Mendham; Jiayun Pang; Martin J. Snowden; Dennis Douroumis (199-208).
The purpose of the study was to investigate and identify the interactions within solid dispersions of cationic drugs and anionic polymers processed by hot-melt extrusion (HME) technique. Propranolol HCl (PRP) and diphenhydramine HCl (DPD) were used as model cationic active substances while pH sensitive anionic methacrylic acid based methyl methacrylate copolymers Eudragit L100® (L100) and ethyl acrylate copolymer Eudragit L100-55 (Acryl EZE) (L100-55) were used as polymeric carriers. The extrudates were further characterised using various physicochemical characterisation techniques to determine the morphology, the drug state within the polymer matrices and the type of drug–polymer interactions. Molecular modelling predicted the existence of two possible H-bonding types while the X-ray photon spectroscopy (XPS) advanced surface analysis of the extrudates revealed intermolecular ionic interactions between the API amino functional groups and the polymer carboxylic groups through the formation of hydrogen bonding. The magnitude of the intermolecular interactions varied according to the drug–polymer miscibility.
Keywords: Hot-melt extrusion; X-ray photoelectron spectroscopy; Intermolecular interactions;

Formulation and drying of miconazole and itraconazole nanosuspensions by Ana M. Cerdeira; Marco Mazzotti; Bruno Gander (209-220).
Miconazole and itraconazole possess adequate membrane permeability, but only slight water solubility, which limits their bioavailability and antifungal effect. To increase their dissolution rate, the compounds were nanoground by media milling to produce nanosuspensions with mean particle size of approximately 210 nm and stabilized with sodium dodecylsulfate (SDS) in combinations with either cellulose ethers (HPC or HPMC) or poloxamers. During storage for 3 months at 25 °C, HPC/SDS stabilized more efficiently miconazole nanoparticles, while poloxamer 407/SDS performed better with itraconazole nanosuspensions. The stabilizing efficiency of the excipients was explained by physical–chemical drug–excipients interactions. The HPC/SDS-stabilized nanosuspensions were spray-dried or freeze-dried with and without the matrix formers mannitol or microcrystalline cellulose (MCC). In absence of matrix former, itraconazole particles agglomerated more extensively than miconazole particles, resulting in a low dissolution rate. Dissolution of the spray- or freeze-dried miconazole nanosuspension was enhanced in presence of mannitol or MCC (drug substance:excipient ratio of 1:1, w/w), as compared to the coarse drug suspension (twice the amount dissolved after 10 and 20 min). Spray-drying itraconazole nanosuspension in presence of mannitol or MCC also yielded fast dissolution (60% dissolved in less than 10 min as compared to 30–45 min with the coarse suspension). Freeze-dried itraconazole nanosuspensions did generally not dissolve substantially faster than freeze-dried coarse suspension. In conclusion, we were able to process miconazole and itraconazole successfully and under similar conditions into dry nanoparticulate drug products with enhanced in vitro performance.
Keywords: Nanosuspension; Nanogrinding; Nanonization; Drug solubility; Miconazole; Itraconazole; Spray-drying; Freeze-drying;

siRNA delivery into tumor cells by lipid-based nanoparticles composed of hydroxyethylated cholesteryl triamine by Yoshiyuki Hattori; Tsukasa Nakamura; Hiroaki Ohno; Nobutaka Fujii; Yoshie Maitani (221-229).
Previously, we reported that cationic nanoparticles (NP) composed of cholesteryl diamine (OH-Chol, (3S)-N-(2-(2-hydroxyethylamino)ethyl)cholesteryl-3-carboxamide) and Tween 80 could deliver plasmid DNA (pDNA) and small interfering RNA (siRNA) with high transfection efficiency into various tumor cells. In this study, to facilitate the endosomal escape of siRNA transfected by lipid-based nanoparticles, we synthesized new cationic cholesteryl triamine (OH-N-Chol, (3S)-N-(2-(2-(2-hydroxyethylamino)ethylamino)ethyl)cholesteryl-3-carboxamide) with an ethylenimine extension and prepared cationic nanoparticles (NP–N) composed of cholesteryl triamine and Tween 80. Although NP–N/siRNA complex (NP–N nanoplex) after mixing NP–N with siRNA was >350 nm in size, the vortex-mixing during the nanoplex formation decreased it to about 200 nm, which was an injectable size. NP–N nanoplex was mainly internalized by macropinocytosis-mediated endocytosis, as was NP nanoplex, and showed higher gene knockdown efficiency than NP nanoplex in human cervical carcinoma SiHa cells. From these results, cationic nanoparticles composed of OH-N-Chol and Tween 80 may have potential as a gene vector for siRNA transfection to tumor cells.
Keywords: Cationic nanoparticles; siRNA delivery; Transfection; Cholesteryl diamine; Cholesteryl triamine;

Tuning drug loading and release properties of diatom silica microparticles by surface modifications by Manpreet Bariana; Moom Sinn Aw; Mahaveer Kurkuri; Dusan Losic (230-241).
Diatomaceous earth (DE), or diatomite silica microparticles originated from fossilized diatoms are a potential substitute for its silica-based synthetic counterparts to address limitations in conventional drug delivery. This study presents the impact of engineered surface chemistry of DE microparticles on their drug loading and release properties. Surface modifications with four silanes, including 3-aminopropyltriethoxy silane (APTES), methoxy-poly-(ethylene-glycol)-silane (mPEG-silane), 7-octadecyltrichlorosilane (OTS), 3-(glycidyloxypropyl)trimethoxysilane (GPTMS) and two phosphonic acids, namely 2-carboxyethyl-phosphonic acid (2 CEPA) and 16-phosphono-hexadecanoic acid (16 PHA) were explored in order to tune drug loading and release characteristics of water insoluble (indomethacin) and water soluble drugs (gentamicin). Successful grafting of these functional groups with different interfacial properties was confirmed using X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). Thermogravimetric analysis (TGA) was applied to determine the amount of loaded drugs and UV-spectrophotometry to analyse in vitro drug release from modified DE microparticles. Differences in drug release time (13–26 days) and loading capacity (14–24%) were observed depending on functional groups on the surface of DE microparticles. It was found that hydrophilic surfaces, due to the presence of polar carboxyl, amine or hydrolyzed epoxy group, favor extended release of indomethacin, while the hydrophobic DE surface modified by organic hydrocarbons gives a better sustained release profile for gentamicin. This work demonstrates that by changing surface functionalities on DE microparticles, it is possible to tune their drug loading and release characteristics for both hydrophobic and hydrophilic drugs and therefore achieve optimal drug delivery performance.
Keywords: Diatomite; Diatomaceous earth; Porous materials; Surface modifications; Drug delivery; Indomethacin; Gentamicin;

Keywords: Stratified medicine; Innovation; Higher education; Financial crisis; Play;

Functional characterization and molecular expression of large neutral amino acid transporter (LAT1) in human prostate cancer cells by Mitesh Patel; Pranjali Dalvi; Mitan Gokulgandhi; Susamita Kesh; Tanvi Kohli; Dhananjay Pal; Ashim K. Mitra (245-253).
The primary objective of this study is to functionally characterize and provide molecular evidence of large neutral amino acid transporter (LAT1) in human derived prostate cancer cells (PC-3). We carried out the uptake of [3H]-tyrosine to assess the functional activity of LAT1. Reverse transcription-polymerase chain reaction (RT-PCR) analysis is carried out to confirm the molecular expression of LAT1. [3H]-tyrosine uptake is found to be time dependent and linear up to 60 min. The uptake process does not exhibit any dependence on sodium ions, pH and energy. However, it is temperature dependent and found maximal at physiological temperature. The uptake of [3H]-tyrosine demonstrates saturable kinetics with K m and V max values of 34 ± 3 μM and 0.70 ± 0.02 nanomoles/min/mg protein, respectively. It is strongly inhibited by large neutral (phenylalanine, tryptophan, leucine, isoleucine) and small neutral (alanine, serine, cysteine) but not by basic (lysine and arginine) and acidic (aspartic and glutamic acid) amino acids. Isoleucine–quinidine (Ile-quinidine) prodrug generates a significant inhibitory effect on [3H]-tyrosine uptake suggesting that it is recognized by LAT1. RT-PCR analysis provided a product band at 658 and 840 bp, specific to LAT1 and LAT2, respectively. For the first time, this study demonstrates that LAT1, primarily responsible for the uptake of large neutral amino acids, is functionally active in PC-3 cells. Significant increase in the uptake generated by Ile-quinidine relative to quinidine suggests that LAT1 can be utilized for enhancing the cellular permeation of poor cell permeable anticancer drugs. Furthermore, this cell line can be utilized as an excellent in vitro model for studying the interaction of large neutral amino acid conjugated drugs with LAT1 transporter.
Keywords: Tyrosine; LAT1; Cellular uptake mechanism; Prostate cancer; Prodrugs; Drug delivery;

A new drug delivery system inhibits uveitis in an animal model after cataract surgery by Simone Eperon; Marta Rodriguez-Aller; Konstantinos Balaskas; Robert Gurny; Yan Guex-Crosier (254-261).
Cataract surgery is a common ocular surgical procedure consisting in the implantation of an artificial intraocular lens (IOL) to replace the ageing, dystrophic or damaged natural one. The management of postoperative ocular inflammation is a major challenge especially in the context of pre-existing uveitis. The association of the implanted IOL with a drug delivery system (DDS) allows the prolonged intraocular release of anti-inflammatory agents after surgery. Thus IOL-DDS represents an “all in one” strategy that simultaneously addresses both cataract and inflammation issues. Polymeric DDS loaded with two model anti-inflammatory drugs (triamcinolone acetonide (TA) and cyclosporine A (CsA)) were manufactured in a novel way and tested regarding their efficiency for the management of intraocular inflammation during the 3 months following surgery. The study involved an experimentally induced uveitis in rabbits. Experimental results showed that medicated DDS efficiently reduced ocular inflammation (decrease of protein concentration in aqueous humour, inflammatory cells in aqueous humour and clinical score). Additionally, more than 60% of the loading dose remained in the DDS at the end of the experiment, suggesting that the system could potentially cover longer inflammatory episodes. Thus, IOL-DDS were demonstrated to inhibit intraocular inflammation for at least 3 months after cataract surgery, representing a potential novel approach to cataract surgery in eyes with pre-existing uveitis.
Keywords: Cyclosporine A; Triamcinolone acetonide; Polymeric drug delivery system; Ocular inflammation; Rabbit; Uveitis;

Encapsulation of Acyclovir in new carboxylated cyclodextrin-based nanosponges improves the agent's antiviral efficacy by David Lembo; Shankar Swaminathan; Manuela Donalisio; Andrea Civra; Linda Pastero; Dino Aquilano; Pradeep Vavia; Francesco Trotta; Roberta Cavalli (262-272).
Cyclodextrin-based nanosponges (NS) are solid nanoparticles, obtained from the cross-linking of cyclodextrins that have been proposed as delivery systems for many types of drugs. Various NS derivatives are currently under investigation in order that their properties might be tuned for different applications. In this work, new carboxylated cyclodextrin-based nanosponges (Carb-NS) carrying carboxylic groups within their structure were purposely designed as novel Acyclovir carriers. TEM measurements revealed their spherical shape and size of about 400 nm. The behaviour of Carb-NS, with respect to the incorporation and delivery of Acyclovir, was compared to that of NS, previously investigated as a drug carrier. DSC, XRPD and FTIR analyses were used to investigate the two NS formulations. The results confirm the incorporation of the drug into the NS structure and NS-Acyclovir interactions. The Acyclovir loading into Carb-NS was higher than that obtained using NS, reaching about 70% (w/w). In vitro release studies showed the release kinetics of Acyclovir from Carb-NS to be prolonged in comparison with those observed with NS, with no initial burst effect. The NS uptake into cells was evaluated using fluorescent Carb-NS and revealed the nanoparticle internalisation. Enhanced antiviral activity against a clinical isolate of HSV-1 was obtained using Acyclovir loaded in Carb-NS.
Keywords: Acyclovir; Nanosponges; Cyclodextrin; Prolonged release; Antiviral activity;

Imidazolium ionic liquids as solvents of pharmaceuticals: Influence on HSA binding and partition coefficient of nimesulide by Ana M.O. Azevedo; Diogo M.G. Ribeiro; Paula C.A.G. Pinto; Marlene Lúcio; Salette Reis; M. Lúcia M.F.S. Saraiva (273-278).
In this work, the influence of imidazolium ionic liquids (ILs) on bio-chemical parameters that influence the in vivo behavior of nimesulide was evaluated. In this context, the binding of nimesulide to human serum albumin (HSA), in IL media, was studied. In parallel, the evaluation of the interaction of drug–IL systems, with micelles of hexadecylphosphocholine (HDPC), enabled the calculation of partition coefficients (K p). Both assays were performed in buffered media in the absence and in the presence of emim [BF4], emim [Ms] and emim [TfMs] 1%.Even though there was an increase of the dissociation constant (K d) in IL media, nimesulide still binds to HSA by means of strong interactions. The thermodynamic analysis indicates that the interaction is spontaneous for all the tested systems. Moreover, the studied systems exhibited properties that are favorable to the interaction of the drug with biological membranes, with K p values 2.5–3.5 higher than in aqueous environment.The studied nimesulide–IL systems presented promising characteristics regarding the absorption and distribution of the drug in vivo, so that the studied solvents seem to be good options for drug delivery.
Keywords: Ionic liquids; Drug solvent/carrier; Drug–protein binding; Partition coefficient;

d-Alpha-tocopherol acid polyethylene glycol 1000 succinate, an effective stabilizer during solidification transformation of baicalin nanosuspensions by Peng-Fei Yue; Jing Wan; Yong Wang; Yu Li; Yue-Qin Ma; Ming Yang; Peng-Yi Hu; Hai-Long Yuan; Chang-Hong Wang (279-287).
Baicalin nanosuspensions, stabilized with 10% TPGS (relative to the weight of baicalin), were transformed into nanosuspensions powders by solidification process. Solidification methods for this transformation included freeze-drying, spray drying or vacuum drying. High pressure homogenization was applied for production of baicalin nanosuspensions used TPGS, SDS, P188, HPMC and MC as stabilizer, respectively. The influence of the different solidification transformation methods on the redispersibility of solid drug nanosuspensions was systemically investigated, such as freeze-drying, spray drying and vacuum drying. Each method was applied with three grades of process stresses called as “conservative”, “moderate” and “aggressive” conditions, and the redispersibility index (RDI) of nanosuspensions stabilized by stabilizers (such as TPGS, SDS, P188, HPMC and MC) during those process was investigated. The results showed that there was significant difference in RDI of nanosuspensions after solidification process. The RDIa (1.09, 1.01, 1.05, 0.99), RDIb (1.03, 0.99, 1.06, 1.02) and RDIc (1.01, 1.01, 1.09, 1.08) of nanosuspensions stabilized by TPGS were more small during different solidification process, compared with those of nanosuspensions stabilized by other stabilizer. It was concluded that the baicalin nanosuspensions were subjected to agglomeration or crystal growth during solidification transformation, especially at high aggressive stress conditions. Meanwhile, compared to other stabilizer, the TPGS was more effective for stability of baicalin nanosuspensions, which could exhibit higher affinity to the drug crystal and stronger surface adsorption at different solidification stresses.
Keywords: Baicalin nanosuspensions; Redispersibility; Solidification; Transformation stress; TPGS; Stabilizer;

Skin accumulation and penetration of a hydrophilic compound by a novel gemini surfactant, sodium dilauramidoglutamide lysine by Tomohiro Hikima; Yoshinaga Tamura; Yukio Yamawaki; Masashi Yamamoto; Kakuji Tojo (288-292).
We investigated a novel peptide-based gemini amphiphilic compound, sodium dilauramidoglutamide lysine (DLGL), as a chemical enhancer for the skin penetration of l-ascorbic acid 2-glucoside (AAG). A three-dimensional cultured human skin product, TESTSKIN™ LSE-high (LSE-high), was used as a skin model. The penetration flux of AAG with DLGL and that obtained with sodium lauramidoglutamide (LG) as a conventional surfactant across LSE-high were increased by 12.56 and 69.29 times compared to the control, respectively. The ratio of AAG amount with DLGL in the skin (21.78% total dose) was significantly increased (p  < 0.05) compared to the control (7.23%) and to the AAG amount with LG (8.13%). The AAG amounts in receptor were 1.06% (control), 3.19% (+DLGL) and 21.00% (+LG). Thus, DLGL preserved AAG in skin, resulting in enhanced AAG penetration flux. However, LG might create the pathways through the skin. We conclude that DLGL is a gemini surfactant that accumulates a hydrophilic compound in skin and enhances the penetration flux. DLGL may therefore be a novel addition agent for skin local therapy.
Keywords: Gemini amphiphilic compound; Sodium dilauramidoglutamide lysine; Peptide; Chemical enhancer;

(1) Pseudoternary phase diagram; (2) drug-loaded NE; (3) gellan gum solution; (4) drug-loaded in situ NE gel; (5) gamma sterilization; (6) histopathological assessment of rabbit's ocular tissues; cornea and (7) aqueous humor concentration–time profiles of T-HCl following ocular instillation of F31 in situ NE gel and the oily drug solution in rabbits (mean ± SD, n  = 5).Terbinafine hydrochloride (T-HCl) is recommended for the management of fungal keratitis. To maintain effective aqueous humor concentrations, frequent instillation of T-HCl drops is necessary. This work aimed to develop alternative controlled-release in situ ocular drug-loaded nanoemulsion (NE) gels. Twelve pseudoternary-phase diagrams were constructed using oils (isopropyl myristate/Miglyol® 812), surfactants (Tween® 80/Cremophor® EL), a co-surfactant (polyethylene glycol 400) and water. Eight drug-loaded (0.5%, w/v) NEs were evaluated for thermodynamic stability, morphology, droplet size and drug release in simulated tear fluid (pH 7.4). Following dispersion in gellan gum solution (0.2%, w/w), the in situ NE gels were characterized for transparency, rheological behavior, mucoadhesive force, drug release and histopathological assessment of ocular irritation. Drug pharmacokinetics of sterilized F31 [Miglyol® 812, Cremophor® EL: polyethylene glycol 400 (1:2) and water (5, 55 and 40%, w/w, respectively)] in situ NE gel and oily drug solution were evaluated in rabbit aqueous humor. The NEs were thermodynamically stable and have spherical droplets (<30 nm). The gels were transparent, pseudoplastic, mucoadhesive and showed more retarded zero-order drug release rates. F31 in situ NE gel showed the least ocular irritation potential and significantly (P  < 0.01) higher C max, delayed T max, prolonged mean residence time and increased bioavailability.
Keywords: Terbinafine hydrochloride; Gellan gum; In situ gels; Nanoemulsions; Aqueous humor;

Transdermal drug delivery of paroxetine through lipid-vesicular formulation to augment its bioavailability by Mohamed A. El-Nabarawi; Ehab R. Bendas; Randa Tag A. El Rehem; Mohammed Y.S. Abary (307-317).
Paroxetine (PAX) is the most potent serotonin reuptake blocker antidepressant clinically available. This study is aimed to reduce the side effects accompanied with the initial high plasma concentration after oral administration of PAX and fluctuations in plasma levels and also to decrease the broad metabolism of the drug in the liver by developing and optimizing liposomal transdermal formulation of PAX in order to improve its bioavailability. PAX liposomes were prepared by reverse phase evaporation technique using lecithin phosphatidylcholine (LPC), cholesterol (CHOL) and drug in different molar ratios. The prepared liposomes were characterized for size, shape, entrapment efficiency and in vitro drug release. The studies demonstrated successful preparation of PAX liposomes. The effect of using different molar ratios of (LPC:CHOL) on entrapment efficiency and on drug release was studied. Liposomes showed percentage entrapment efficiency (%EE) of 81.22 ± 3.08% for optimized formula (F5) which composed of (LPC:CHOL, 7:7) and 20 mg of PAX, with average vesicle size of 220.53 ± 0.757 nm. The selected formula F5 (7:7) was incorporated in gel bases of HPMC-E4M (2%, 4%, and 6%). The selected formula of PAX liposomal gel of HPMC-E4M (2% and 4%) were fabricated in the reservoir type of transdermal patches and evaluated through in vitro release. After that the selected formula of PAX liposomal gel transdermal patch was applied to rabbits for in vivo bioavailability study in comparison with oral administration of the marketed PAX tablet.An HPLC method was developed for the determination of PAX in plasma of rabbits after transdermal patch application and oral administration of the marketed PAX tablets of 20 mg dose. The intra- and inter-day accuracy and precision were determined as relative error and relative standard deviation, respectively. The linearity was assessed in the range of 5–200 ng/ml.Pharmacokinetic parameters were determined as the C max of PAX liposomal transdermal patch was found to be 92.53 ng/ml at t max of 12 h and AUC0–48 was 2305.656 ng h/ml and AUC0–∞ was 3852.726 ng h/ml, compared to the C max of 172.35 ng/ml after oral administration of the marketed PAX tablet with t max of 6 h and AUC0–24 was 1206.63 ng h/ml and AUC0–∞ was 1322.878 ng h/ml. These results indicate improvement of bioavailability of the PAX after liposomal transdermal patch application and sustaining of the therapeutic effects compared to oral administration.
Keywords: Liposomes; Paroxetine; Lecithin phosphatidylcholine; Cholesterol; In vitro drug release; Liposomal gel; Transdermal patch; HPLC assay method; Bioavailability study;

Clarithromycin-loaded liposomes offering high drug loading and less irritation by Xiaona Liu; Wei Sun; Bo Zhang; Bin Tian; Xing Tang; Na Qi; Haibing He; Huifang Li; Xiangqun Jin (318-327).
The aim of this study was to develop an efficient method of preparing less irritant clarithromycin-loaded liposomes (CLA-Lip) for injection with a high drug loading and to evaluate their physicochemical characteristics before and after lyophilization. CLA-Lip were prepared using the film-dispersion method with sodium cholesterol sulfate (SCS) and n-hexyl acid as the regulators and then lyophilized. The liposomes were characterized in terms of their size, size distribution, zeta potential, morphology, in vitro release, haemolysis, and lyophilization and irritation testing was carried out. The TEM images revealed that the structure of the CLA-Lip were multilamellar and of a regular size of around 100 nm. In addition, the lyophilized CLA-Lip were characterized by DSC and Infrared spectroscopy to confirm the structure. H-bonding and salt-forming reactions were used to ensure that clarithromycin (CLA) was stably encapsulated in the liposomes. This method provided a 30-fold increase in the concentration of clarithromycin relative to that in aqueous solution. Sucrose was found to be the best protective agent and was added in an amount of 12.5% (w/v). According to the mouse scratch test and the rat paw lick test, the pain of CLA-Lip was significantly reduce by approximately 80% compared with the solution of clarithromycin phosphate. In addition, rabbit ear vein experiments produced similar results. These findings suggested that CLA-Lip was a stable delivery system with less irritation, which should be extremely suitable for clinical application.
Keywords: Clarithromycin; N-hexanoic; Multilamellar liposomes; Lyophilization; Less irritation;